Thalamocortical organoids enable in vitro modeling of 22q11.2 microdeletion associated with neuropsychiatric disorders.

Thalamic dysfunction has been implicated in multiple psychiatric disorders. We sought to study the mechanisms by which abnormalities emerge in the context of the 22q11.2 microdeletion, which confers significant genetic risk for psychiatric disorders. We investigated early stages of human thalamus development using human pluripotent stem cell-derived organoids and show that the 22q11.2 microdeletion underlies widespread transcriptional dysregulation associated with psychiatric disorders in thalamic neurons and glia, including elevated expression of FOXP2. Using an organoid co-culture model, we demonstrate that the 22q11.2 microdeletion mediates an overgrowth of thalamic axons in a FOXP2-dependent manner. Finally, we identify ROBO2 as a candidate molecular mediator of the effects of FOXP2 overexpression on thalamic axon overgrowth. Together, our study suggests that early steps in thalamic development are dysregulated in a model of genetic risk for schizophrenia and contribute to neural phenotypes in 22q11.2 deletion syndrome.

Context-invariant socioemotional encoding by prefrontal ensembles.

The prefrontal cortex plays a key role in social interactions, anxiety-related avoidance, and flexible context- dependent behaviors, raising the question: how do prefrontal neurons represent socioemotional information across different environments? Are contextual and socioemotional representations segregated or intermixed, and does this cause socioemotional encoding to remap or generalize across environments? To address this, we imaged neuronal activity in the medial prefrontal cortex of mice engaged in social interactions or anxiety-related avoidance within different environments. Neuronal ensembles representing context and social interaction overlapped more than expected while remaining orthogonal. Anxiety-related representations similarly generalized across environments while remaining orthogonal to contextual information. This shows how prefrontal cortex multiplexes parallel information streams using the same neurons, rather than distinct subcircuits, achieving context-invariant encoding despite context-specific reorganization of population-level activity.

Distinct transcriptional programs define a heterogeneous neuronal ensemble for social interaction.

Reliable representations of information regarding complex behaviors including social interactions require the coordinated activity of heterogeneous cell types within distributed brain regions. Activity in the medial prefrontal cortex is critical in regulating social behavior, but our understanding of the specific cell types which comprise the social ensemble has been limited by available mouse lines and molecular tagging strategies which rely on the expression of a single marker gene. Here we sought to quantify the heterogeneous neuronal populations which are recruited during social interaction in parallel in a non-biased manner and determine how distinct cell types are differentially active during social interactions. We identify distinct populations of prefrontal neurons activated by social interaction by quantification of immediate early gene (IEG) expression in transcriptomically clustered neurons. This approach revealed variability in the recruitment of different excitatory and inhibitory populations within the medial prefrontal cortex. Furthermore, evaluation of the populations of IEGs recruited following social interaction revealed both cell-type and region-specific transcriptional programs, suggesting that reliance on a single molecular marker is insufficient to quantify activation across all cell types. Our findings provide a comprehensive description of cell-type specific transcriptional programs invoked by social interactions and reveal new insights into the heterogeneous neuronal populations which compose the social ensemble.

Dynamic patterns of correlated activity in the prefrontal cortex encode information about social behavior.

New technologies make it possible to measure activity from many neurons simultaneously. One approach is to analyze simultaneously recorded neurons individually, then group together neurons which increase their activity during similar behaviors into an "ensemble." However, this notion of an ensemble ignores the ability of neurons to act collectively and encode and transmit information in ways that are not reflected by their individual activity levels. We used microendoscopic GCaMP imaging to measure prefrontal activity while mice were either alone or engaged in social interaction. We developed an approach that combines a neural network classifier and surrogate (shuffled) datasets to characterize how neurons synergistically transmit information about social behavior. Notably, unlike optimal linear classifiers, a neural network classifier with a single linear hidden layer can discriminate network states which differ solely in patterns of coactivity, and not in the activity levels of individual neurons. Using this approach, we found that surrogate datasets which preserve behaviorally specific patterns of coactivity (correlations) outperform those which preserve behaviorally driven changes in activity levels but not correlated activity. Thus, social behavior elicits increases in correlated activity that are not explained simply by the activity levels of the underlying neurons, and prefrontal neurons act collectively to transmit information about socialization via these correlations. Notably, this ability of correlated activity to enhance the information transmitted by neuronal ensembles is diminished in mice lacking the autism-associated gene Shank3. These results show that synergy is an important concept for the coding of social behavior which can be disrupted in disease states, reveal a specific mechanism underlying this synergy (social behavior increases correlated activity within specific ensembles), and outline methods for studying how neurons within an ensemble can work together to encode information.

Shank-cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses.

The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function.

Multiple spatial and kinetic subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking PALM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is essential for synaptic plasticity underlying memory formation. Some functions of CaMKII are mediated by interactions with synaptic proteins, and activity-triggered translocation of CaMKII to synapses has been heavily studied. However, CaMKII actions away from the postsynaptic density (PSD) remain poorly understood, in part because of the difficulty in discerning where CaMKII binds in live cells. We used photoactivated localization microscopy (PALM) in rat hippocampal neurons to track single molecules of CaMKIIα, mapping its spatial and kinetic heterogeneity at high resolution. We found that CaMKIIα exhibits at least three kinetic subpopulations, even within individual spines. Latrunculin application or coexpression of CaMKIIß carrying its actin-binding domain strongly modulated CaMKII diffusion, indicating that a major subpopulation is regulated by the actin cytoskeleton. CaMKII in spines was typically more slowly mobile than in dendrites, consistent with presence of a higher density of binding partners or obstacles. Importantly, NMDA receptor stimulation that triggered CaMKII activation prompted the immobilization and presumed binding of CaMKII in spines not only at PSDs but also at other points up to several hundred nanometers away, suggesting that activated kinase does not target only the PSD. Consistent with this, single endogenous activated CaMKII molecules detected via STORM immunocytochemistry were concentrated in spines both at the PSD and at points quite distant from the synapse. Together, these results indicate that CaMKII mobility within spines is determined by association with multiple interacting proteins, even outside the PSD, suggesting diverse mechanisms by which CaMKII may regulate synaptic transmission.

Specific sorting and post-Golgi trafficking of dendritic potassium channels in living neurons.

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.

Temporal phase mask encrypted optical steganography carried by amplified spontaneous emission noise.

A temporal phase mask encryption method is proposed and experimentally demonstrated to improve the security of the stealth channel in an optical steganography system. The stealth channel is protected in two levels. In the first level, the data is carried by amplified spontaneous emission (ASE) noise, which cannot be detected in either the time domain or spectral domain. In the second level, even if the eavesdropper suspects the existence of the stealth channel, each data bit is covered by a fast changing phase mask. The phase mask code is always combined with the wide band noise from ASE. Without knowing the right phase mask code to recover the stealth data, the eavesdropper can only receive the noise like signal with randomized phase.

Optimization of cell morphology measurement via single-molecule tracking PALM.

In neurons, the shape of dendritic spines relates to synapse function, which is rapidly altered during experience-dependent neural plasticity. The small size of spines makes detailed measurement of their morphology in living cells best suited to super-resolution imaging techniques. The distribution of molecular positions mapped via live-cell Photoactivated Localization Microscopy (PALM) is a powerful approach, but molecular motion complicates this analysis and can degrade overall resolution of the morphological reconstruction. Nevertheless, the motion is of additional interest because tracking single molecules provides diffusion coefficients, bound fraction, and other key functional parameters. We used Monte Carlo simulations to examine features of single-molecule tracking of practical utility for the simultaneous determination of cell morphology. We find that the accuracy of determining both distance and angle of motion depend heavily on the precision with which molecules are localized. Strikingly, diffusion within a bounded region resulted in an inward bias of localizations away from the edges, inaccurately reflecting the region structure. This inward bias additionally resulted in a counterintuitive reduction of measured diffusion coefficient for fast-moving molecules; this effect was accentuated by the long camera exposures typically used in single-molecule tracking. Thus, accurate determination of cell morphology from rapidly moving molecules requires the use of short integration times within each image to minimize artifacts caused by motion during image acquisition. Sequential imaging of neuronal processes using excitation pulses of either 2 ms or 10 ms within imaging frames confirmed this: processes appeared erroneously thinner when imaged using the longer excitation pulse. Using this pulsed excitation approach, we show that PALM can be used to image spine and spine neck morphology in living neurons. These results clarify a number of issues involved in interpretation of single-molecule data in living cells and provide a method to minimize artifacts in single-molecule experiments.

Outer membrane targeting, ultrastructure, and single molecule localization of the enteropathogenic Escherichia coli type IV pilus secretin BfpB.

Type IV pili (T4P) are filamentous surface appendages required for tissue adherence, motility, aggregation, and transformation in a wide array of bacteria and archaea. The bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is a prototypical T4P and confirmed virulence factor. T4P fibers are assembled by a complex biogenesis machine that extrudes pili through an outer membrane (OM) pore formed by the secretin protein. Secretins constitute a superfamily of proteins that assemble into multimers and support the transport of macromolecules by four evolutionarily ancient secretion systems: T4P, type II secretion, type III secretion, and phage assembly. Here, we determine that the lipoprotein transport pathway is not required for targeting the BfpB secretin protein of the EPEC T4P to the OM and describe the ultrastructure of the single particle averaged structures of the assembled complex by transmission electron microscopy. Furthermore, we use photoactivated localization microscopy to determine the distribution of single BfpB molecules fused to photoactivated mCherry. In contrast to findings in other T4P systems, we found that BFP components predominantly have an uneven distribution through the cell envelope and are only found at one or both poles in a minority of cells. In addition, we report that concurrent mutation of both the T4bP secretin and the retraction ATPase can result in viable cells and found that these cells display paradoxically low levels of cell envelope stress response activity. These results imply that secretins can direct their own targeting, have complex distributions and provide feedback information on the state of pilus biogenesis.

A network of networks: cytoskeletal control of compartmentalized function within dendritic spines.

Almost 30 years ago, actin was identified as the major cytoskeletal component of dendritic spines. Since then, its role in the remarkable dynamics of spine morphology have been detailed with live-cell views establishing that spine shape dynamics are an important requirement for synaptogenesis and synaptic plasticity. However, the actin cytoskeleton is critical to numerous and varied processes within the spine which contribute to the maintenance and plasticity of synaptic function. Here, we argue that the spatial and temporal distribution of actin-dependent processes within spines suggests that the spine cytoskeleton should not be considered a single entity, but an interacting network of nodes or hubs that are independently regulated and balanced to maintain synapse function. Disruptions of this balance within the spine are likely to lead to psychiatric and neurological dysfunction.

Single-molecule discrimination of discrete perisynaptic and distributed sites of actin filament assembly within dendritic spines.

Within dendritic spines, actin is presumed to anchor receptors in the postsynaptic density and play numerous roles regulating synaptic transmission. However, the submicron dimensions of spines have hindered examination of actin dynamics within them and prevented live-cell discrimination of perisynaptic actin filaments. Using photoactivated localization microscopy, we measured movement of individual actin molecules within living spines. Velocity of single actin molecules along filaments, an index of filament polymerization rate, was highly heterogeneous within individual spines. Most strikingly, molecular velocity was elevated in discrete, well-separated foci occurring not principally at the spine tip, but in subdomains throughout the spine, including the neck. Whereas actin velocity on filaments at the synapse was substantially elevated, at the endocytic zone there was no enhanced polymerization activity. We conclude that actin subserves spatially diverse, independently regulated processes throughout spines. Perisynaptic actin forms a uniquely dynamic structure well suited for direct, active regulation of the synapse.

PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP.

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was approximately 17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

Activator protein-1 activity regulates epithelial tumor cell identity.

To examine the consequences of inhibiting activator protein-1 (AP-1) transcription factors in skin, transgenic mice were generated, which use the tetracycline system to conditionally express A-FOS, a dominant negative that inhibits AP-1 DNA binding. Older mice develop mild alopecia and hyperplasia of sebaceous glands, particularly around the eyes. When A-FOS was expressed during chemical-induced skin carcinogenesis, mice do not develop characteristic benign and malignant squamous lesions but instead develop benign sebaceous adenomas containing a signature mutation in the H-ras proto-oncogene. Inhibiting AP-1 activity after tumor formation caused squamous tumors to transdifferentiate into sebaceous tumors. Furthermore, reactivating AP-1 in sebaceous tumors results in a reciprocal transdifferentiation into squamous tumors. In both cases of transdifferentiation, individual cells express molecular markers for both cell types, indicating individual tumor cells have the capacity to express multiple lineages. Molecular characterization of cultured keratinocytes and tumor material indicates that AP-1 regulates the balance between the wnt/beta-catenin and hedgehog signaling pathways that determine squamous and sebaceous lineages, respectively. Chromatin immunoprecipitation analysis indicates that c-Jun binds several wnt promoters, which are misregulated by A-FOS expression, suggesting that members of the wnt pathway can be a primary targets of AP-1 transcriptional regulation. Thus, AP-1 activity regulates tumor cell lineage and is essential to maintain the squamous tumor cell identity.